Presentation on theme: "SUPPLEMENTARY FIGURES Figure S-I. Fluorescence intensity and subcellular localization of transfected EGFP fusion proteins HeLa cells were transferred into."— Presentation transcript:
SUPPLEMENTARY FIGURES Figure S-I. Fluorescence intensity and subcellular localization of transfected EGFP fusion proteins HeLa cells were transferred into 96-well plates (Costar) at 5 x 10 3 /well each, incubated for 24 h and then transfected with EGFP, EGFP-importin 2 full-length and EGFP-importin 2 C-mutant constructs using Effectene Transfection Regent (Qiagen). To quantitate cytoplasmic and nuclear fluorescence, images were captured on an ArrayScan VTI instrument (Thermo Fisher) and analysed using Cellomics software and the Compartmental Analysis Bioapplication. The analysis algorithm uses DAPI fluorescence to define a mask used to measure nuclear fluorescence. The region for whole cell measurements is generated by dilating the mask to cover as much of the cytoplasm as possible without going outside the cell boundary. The cytoplasm region is generated by removal of the nuclear from whole cell regions to create a ring. Average pixel intensities of EGFP within these regions were calculated. Non-transfected cells (with autofluorescent pixel intensity similar to non-transfected controls) were gated out of the analysis. Three independent experiments were done, and 300 cells were measured for each transfection. One of the independent results was presented here. Figure S-II. Validation of down-regulated genes by quantitative PCR HeLa cells were transfected with EGFP as a control, EGFP-importin 2 full-length or C-mutant constructs, and mRNA expression levels of indicated genes were analyzed by qPCR. The results were from three independent experiments and were presented in comparison to values in the EGFP-expressing cells as the mean ± SEM (n=3 each). **p<0.01; Student s t-test. Primers are shown in Supplemental Table S-III. Figure S-III. Validation of ChIP analysis (A) HeLa cells were exposed to 200 μM H 2 O 2 for 30 min and subjected to immunoprecipitation using an anti-importin 3 antibody in accordance with ChIP assay. The bound proteins were analyzed by Western blotting using anti-importin 3 antibody. (B) HeLa cells were exposed to 200 μM H 2 O 2 for 30 min and subjected to immunoprecipitation using an anti- p53 antibody. After DNA purification from the precipitated the chromatin sample, PCR was performed to amplify sequences in the indicated promoter regions. Figure S-IV. Establishment of STK35 expressing stable cell lines and knock-down experiments by RNAi oligonucleotides (A) 293F cells were transfected with wtag, wtag-STK35S or wtag-STK35L1 and established stable cell lines were selected by single cell clones. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS-PAGE and detected by Western blotting using anti-GST antibody. The band provided with asterisk is suspected to be a degradation product of STK35L1. (B) 293F stably expressing wtag as a control (Cont.), wtag-STK35S or wtag-STK35L1 were pretreated for 30 min in the absence or presence of 50 μM zVAD-fmk and exposed to 2 mM H 2 O 2 for 2 h or 0.5 μM STS for 24 h, respectively. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS- PAGE and analyzed by Western blotting for PARP, and GAPDH. (C) 293F cells stably expressing wtag as a control (Cont.), wtag-STK35S or wtag-STK35L1 were exposed to 0.5 μM STS for 4 h. Cells were stained with PI and then sorted by FACS. Values are means ± SEM (n=3 each) of PI-positive cells. (D) HeLa cells were transfected with Luciferase siRNA (siLUC.) as a control or STK35 siRNA oligonucleotides (siSTK35-1 or siSTK35-2) and incubated for 48 h. The mRNA expression levels of STK35 were analyzed by qPCR. The results are from three independent experiments and are presented in comparison to the values in control siRNA-expressing cells as the mean ±SEM (n=3 each). **p<0.01; Student s t-test.
EGFP-importin 2 Full EGFP-importin 2 C-mut 4 Total intensity (log 10 ) 0 10 20 564 N/C ratio Total intensity (log 10 ) 0 10 20 564 Total intensity (log 10 ) 0 10 20 56 EGFP Figure S-I N/C ratio
Input Normal IgG (Rabbit) Anti-p53 p21 STK35 B Importin 3 IgG heavy chain kDa 30 40 50 60 80 Input Normal IgG (Goat) Anti-importin 3 A Figure S-III
Cleaved PARP GAPDH Cont.STK35SSTK35L1 Untreated H2O2H2O2 H 2 O 2 + z-VAD-fmk STS + z-VAD-fmk Cont.STK35SSTK35L1Cont.STK35SSTK35L1Cont.STK35SSTK35L1Cont.STK35SSTK35L1 B Figure S-IV % PI positive cells Cont. STK35S STK35L1 STSUntreated 0 10 20 30 40 50 60 70 80 C wtag wtag-STK35S wtag-STK35L1 kDa 20 30 40 50 60 80 * A Relative STK35 mRNA level 0 0.2 0.4 0.6 0.8 1.0 1.2 ** siSTK35-2 ** siSTK35-1 siLUC. D
a) Fold change ( or 2-fold) indicates the ratio of microarray signals in the EGFP-importin 2 full-length gene expressing cells compared to those in the control EGFP transfected cells. b) Fold change ( or 2-fold) indicates the ratio of microarray signals of the EGFP-importin 2 C-mutant gene expressing cells to those of the control EGFP transfected cells. c) Partial molecular function/Characterization is provided according to the PANTHER classification system (http://www.pantherdb.org/) d) Abbreviations; N: Nucleus, C: Cytoplasm, M: Membrane, NM: Nuclear Membrane, PM: Plasma Membrane, Ce: Centrosom, Mit: Mitochondria, S: Secreted, G: Golgi, ER: Endoplasmic Reticulum, AJ: Adherents Junctions, CG: Cytoplasmic granular Table S-I. Summary of up- or down-regulated genes in microarray analysis
Table S-II. Summary of replication-dependent histone genes
Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val FullUp-regulated None 21-0.195-0.4970.995 Down-regulated GCM_NF2 C-mut 210.2670.7530.812 Up-regulated GCM_NF2 Down-regulated None Table S-IV. GSEA tests for enrichment of MSigDB C4 gene sets Gene Set Name a) Size b) ES c) NES d) NOM p-val e) FDR q-val FullUp-regulated None Down-regulated None C-mutUp-regulated None Down-regulated None Table S-IV. GSEA tests for enrichment of MSigDB C5 gene sets